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J Lebowitz

Publications and source records attributed to J Lebowitz.

At least 19 recordsLinked to original sources
PubMed2001

Oligomeric structure of virion-associated and soluble forms of the simian immunodeficiency virus envelope protein in the prefusion activated conformation.

The envelope proteins (env) of simian immunodeficiency virus (SIV) and HIV type 1 assemble to form noncovalently associated oligomers in the endoplasmic reticulum. After cleavage in a Golgi compartment, oligomeric env complexes are transported to the surface of infected cells, where incorporation into budding virions can occur. Difficulties in obtaining adequate quantities of virions retaining env, as well as the unstable nature and hydrophobicity of the oligomer, may account for the absence of previous biophysical studies to determine the oligomeric valency of membrane-associated env. The aim of this study was to evaluate the oligomeric state of SIV env before membrane-fusion activation. Virion-associated env, obtained by crosslinking and detergent extraction, and non-crosslinked secreted env ectodomain (recombinant gp140) were purified by lentil-lectin chromatography and gel filtration as single predominant species. Sedimentation equilibrium-derived mass values for both forms of SIV env were close to those predicted for trimeric assemblies. Determination of the mass of individual molecules by scanning transmission electron microscopy confirmed that SIV virion-associated env and gp140 formed largely homogeneous populations of trimers. Furthermore, a triangular or tri-lobed morphology was clearly visualized in a subset of the trimers.

Biopolymers↗
PubMed2001

SmtB-DNA and protein-protein interactions in the formation of the cyanobacterial metallothionein repression complex: Zn2+ does not dissociate the protein-DNA complex in vitro.

The synechococcal metallothionein locus smt consists of two divergent genes: smtA coding for the metallothionein SmtA, and smtB coding for the trans-acting regulator SmtB. The latter binds at two inverted repeats, designated S1/S2 and S3/S4, in the overlapping promoter/operator sites between the two genes. We have determined the binding stoichiometries to the entire operator/promoter DNA and to the separate S1/S2 and S3/S4 half-operator oligonucleotides using sedimentation equilibrium and sedimentation velocity measurements. The full promoter/operator DNA binds two SmtB dimers. The hydrodynamic behavior of this complex supports a compact nucleoprotein structure. Each separate S1/S2 and S3/S4 operator sequence also binds two dimers. An equal molar mixture of separate S1/S2 and S3/S4 operator sequences, in excess SmtB, forms a S1/S2-SmtB:SmtB-S3/S4 bridge complex. Combining these results with previously published binding interference data, which showed consecutive S1/S2 and S3/S4 SmtB occupancy on the operator/promoter DNA, we have developed a model for the establishment of the repression complex that appears to involve significant DNA compaction, presumably DNA bending, stabilized by SmtB-SmtB bridge interactions. DNase I footprinting titrations also showed consecutive S1/S2 and S3/S4 SmtB occupancy. The footprints expand considerably in the presence of Zn2+. Hence, SmtB remains bound to the operator sites when Zn2+ ions are present. This result is further supported by gel retardation assay. Failure of the metal ions to dissociate SmtB from the DNA points to a hitherto unknown function of SmtB in the regulation of the smt locus.

Amino Acid Sequence↗
PubMed2000

Production and characterization of the functional fragment of pneumococcal surface protein A.

Pneumococcal surface protein A (PspA) is present on the cell wall of Streptococcus pneumoniae pathogen and has an antigenetically variable N-terminal domain. This aminoterminal domain is essential for full pneumococcal virulence, and monoclonal antibodies raised against it protect mice against pneumococcal infections. We have cloned and expressed a 34-kDa N-terminal fragment of PspA in Escherichia coli in a soluble form using the T7 RNA polymerase pET-20b vector system. Nickel chelate affinity purification followed by size exclusion and anion exchange chromatography yielded large amounts of pure and homogeneous protein. Analytical ultracentrifugation sedimentation velocity band and boundary studies showed that the molecule was present in aqueous solutions in a monomeric form with an axial shape ratio of approximately 1:12, typical of fibrous proteins. Sequence analyses indicated an alpha-helical coiled-coil structure for this monomeric molecule with only few loop-type breaks in helicity. The mostly alpha-helical structure of this PspA construct was consistent with circular dichroism spectroscopy data. Based on the ultracentrifugation studies, the circular dichroism spectra, and the PspA's sequence analyses, two structural models for the amino-terminal part of the PspA molecule are proposed. The evident highly charged and polar character of the surface of the modeled structures suggests functional properties of PspA that are related to the prevention of S. pneumoniae interactions with the host complement system.

Amino Acid Sequence↗
PubMed2000

Down-regulation of cell surface receptors is modulated by polar residues within the transmembrane domain.

How recycling receptors are segregated from down-regulated receptors in the endosome is unknown. In previous studies, we demonstrated that substitutions in the transferrin receptor (TR) transmembrane domain (TM) convert the protein from an efficiently recycling receptor to one that is rapidly down regulated. In this study, we demonstrate that the "signal" within the TM necessary and sufficient for down-regulation is Thr(11)Gln(17)Thr(19) (numbering in TM). Transplantation of these polar residues into the wild-type TR promotes receptor down-regulation that can be demonstrated by changes in protein half-life and in receptor recycling. Surprisingly, this modification dramatically increases the TR internalization rate as well ( approximately 79% increase). Sucrose gradient centrifugation and cross-linking studies reveal that propensity of the receptors to self-associate correlates with down-regulation. Interestingly, a number of cell surface proteins that contain TM polar residues are known to be efficiently down-regulated, whereas recycling receptors for low-density lipoprotein and transferrin conspicuously lack these residues. Our data, therefore, suggest a simple model in which specific residues within the TM sequences dramatically influence the fate of membrane proteins after endocytosis, providing an alternative signal for down-regulation of receptor complexes to the well-characterized cytoplasmic tail targeting signals.

Amino Acid Sequence↗
PubMed2000

The human immunodeficiency virus type 1 gp120 V2 domain mediates gp41-independent intersubunit contacts.

The envelope protein of human immunodeficiency virus type 1 HIV-1 undergoes proteolytic cleavage in the Golgi complex to produce subunits designated gp120 and gp41, which remain noncovalently associated. While gp41 has a well-characterized oligomeric structure, the maintenance of gp41-independent gp120 intersubunit contacts remains a contentious issue. Using recombinant vaccinia virus to achieve high-level expression of gp120 in mammalian cells combined with gel filtration analysis, we were able to isolate a discrete oligomeric form of gp120. Oligomerization of gp120 occurred intracellularly between 30 and 120 min after synthesis. Analysis by sedimentation equilibrium unequivocally identified the oligomeric species as a dimer. In order to identify the domains involved in the intersubunit contact, we expressed a series of gp120 proteins lacking various domains and assessed the effects of mutation on oligomeric structure. Deletion of the V1 or V3 loops had little effect on the relative amounts of monomer and dimer in comparison to wild-type gp120. In contrast, deletion of either all or part of the V2 loop drastically reduced dimer formation, indicating that this domain is required for intersubunit contact formation. Consistent with this, the V2 loop of the dimer was less accessible than that of the monomer to a specific monoclonal antibody. Previous studies have shown that while the V2 loop is not an absolute requirement for viral entry, the absence of this domain reduces viral resistance to neutralization by monoclonal antibodies or sera. We propose that the quaternary structure of gp120 may contribute to resistance to neutralization by limiting the exposure of conserved epitopes.

Cell Line↗
PubMed2000

Study of the assembly of vesicular stomatitis virus N protein: role of the P protein.

To derive structural information about the vesicular stomatitis virus (VSV) nucleocapsid (N) protein, the N protein and the VSV phosphoprotein (P protein) were expressed together in Escherichia coli. The N and P proteins formed soluble protein complexes of various molar ratios when coexpressed. The major N/P protein complex was composed of 10 molecules of the N protein, 5 molecules of the P protein, and an RNA. A soluble N protein-RNA oligomer free of the P protein was isolated from the N/P protein-RNA complex using conditions of lowered pH. The molecular weight of the N protein-RNA oligomer, 513,879, as determined by analytical ultracentrifugation, showed that it was composed of 10 molecules of the N protein and an RNA of approximately 90 nucleotides. The N protein-RNA oligomer had the appearance of a disk with outer diameter, inner diameter, and thickness of 148 +/- 10 A, 78 +/- 9 A, and 83 +/- 8 A, respectively, as determined by electron microscopy. RNA in the complexes was protected from RNase digestion and was stable at pH 11. This verified that N/P protein complexes expressed in E. coli were competent for encapsidation. In addition to coexpression with the full-length P protein, the N protein was expressed with the C-terminal 72 amino acids of the P protein. This portion of the P protein was sufficient for binding to the N protein, maintaining it in a soluble state, and for assembly of N protein-RNA oligomers. With the results provided in this report, we propose a model for the assembly of an N/P protein-RNA oligomer.

Cloning, Molecular↗
PubMed2000

Lectures.

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Journal Article↗
PubMed1999

Reexamination of the high mobility group-1 protein for self-association and characterization of hydrodynamic properties.

Previous studies of the 25 kDa high mobility group-1 (HMG-1) protein have generated conflicting results regarding whether HMG-1 exists as a monomer or is capable of oligomerizing to (functional) tetramers. To resolve this question, sedimentation velocity analysis yielded a s20,w value of 2.59S, which is consistent with a monomeric protein. Equilibrium sedimentation data were obtained for three HMG-1 concentrations at two rotor speeds. The six sets of data were fit to both an ideal single component and monomer-dimer equilibrium model, with essentially identical fits produced for both models, with the latter indicating a low extent (7%) of dimerization. Reaction of HMG-1 with glutaraldehyde produced a small population of oligomers consistent with a low level of dimers. This supported the monomer-dimer equilibrium model. Surprisingly, gel permeation chromatography yielded an apparent molecular mass of approx. 55 kDa for both HMG-1 and HMG-2. This finding is considered anomalous and presumably due to the high negative charge density in the C terminus of HMG-1. The sedimentation data also permit one to model HMG-1 as a hydrated prolate ellipsoid with a major axis/minor axis ratio of 2. 79. The collective evidence from the sedimentation and chemical cross-linking studies strongly supports a moderately asymmetric monomer in solution and unequivocally eliminates the possibility of a highly extended shape for HMG-1 or the existence of any extensive oligomerization.

Animals↗
PubMed1999

The integral divalent cation within the intermolecular purine*purine. pyrimidine structure: a variable determinant of the potential for and characteristics of the triple helical association.

In vitro assembly of an intermolecular purine*purine.pyrimidine triple helix requires the presence of a divalent cation. The relationships between cation coordination and triplex assembly were investigated, and we have obtained new evidence for at least three functionally distinct potential modes of divalent cation coordination. (i) The positive influence of the divalent cation on the affinity of the third strand for its specific target correlates with affinity of the cation for coordination to phosphate. (ii) Once assembled, the integrity of the triple helical structure remains dependent upon its divalent cation component. A mode of heterocyclic coordination/chelation is favorable to triplex formation by decreasing the relative tendency for efflux of integral cations from within the triple helical structure. (iii) There is also a detrimental mode of base coordination through which a divalent cation may actively antagonize triplex assembly, even in the presence of other supportive divalent cations. These results demonstrate the considerable impact of the cationic component, and suggest ways in which the triple helical association might be positively or negatively modulated.

Base Pairing↗
PubMed1998

The lipid-free structure of apolipoprotein A-I: effects of amino-terminal deletions.

Deletion mutants of human apolipoprotein A-I (apo hA-I) have been produced from a bacterial expression system to explore the function of the specific domains comprising residues 1-43, 1-65, 88-98, and 187-243, respectively, in the lipid-free conformation and in the lipid-binding mechanism of apo hA-I. Initial studies on apo Delta(1-43)A-I and apo Delta(187-243)A-I have already been reported. To aid purification of these mutants, a histidine-containing N-terminal extension was incorporated (+his); in cases where comparison with the (-his) construct was possible, little effect on the physical properties due to the (+his) extension was found. All mutants have folded structures in their lipid-free state, however these structures differ widely in their relative thermodynamic stability and extent of secondary structure. The mutant with the fewest residues deleted, apo Delta(88-98)A-I(+his), has the least secondary structure (only 34% helix) and is also the least stable (DeltaG = 2.9 kcal/mol). Determined from sedimentation velocity measurements on the lipid-free proteins, all but apo Delta(1-65)A-I(+his) exhibited a range of conformers in solution, which fluctuated around a highly elongated species (dimensions equal to approximately (14-16) x approximately 2.3 nm). Apo Delta(1-65)A-I(+his) exhibited a discrete species which was less asymmetric (dimensions equal to 9 x 2.9 nm). Apo Delta(88-98)A-I(+his) showed extreme heterogeneity with no predominating conformer. Spectroscopic studies (ANS binding and circular dichroism) indicate that there is little difference in the lipid-free structure of the carboxy-terminal deletion mutant, apo Delta(187-243)A-I(+/-his) compared to wild-type (wt) apo wtA-I(+/-his), but substantial differences are observed between wt and the amino-terminal deletion mutants, apo Delta(1-43)A-I, apo Delta(1-65)A-I(+his), and apo Delta(88-98)A-I(+his). In contrast, the lipid-binding properties are impaired for apo Delta(187-243)A-I(+/-his), as measured by dimyristoyl phosphatidylcholine (DMPC) liposome turbidity clearance kinetics and palmitoyloleoyl phosphatidylcholine (POPC) equilibrium binding. Apo Delta(1-43)A-I, apo Delta(1-65)A-I(+his), and apo Delta(88-98)A-I(+his) show lipid affinities statistically similar to apo wtA-I(+his), but significantly defective DMPC clearance kinetics. Interestingly, lecithin:cholesterol acyltransferase (LCAT) activation results correlate qualitatively with the lipid-binding affinity for all mutants but apo Delta(88-98)A-I(+his), suggesting that this mutant has an altered and possibly noncooperative lipid-bound structure as well as an altered lipid-free structure. These results suggest helix 1 (residues 44-65) and helix 10 (residues 220-240) are both required for native lipid-binding properties, while the presence of internal residues, at least helix 3 (residues 88-98), is essential for proper folding of both the lipid-free and lipid-bound conformations. Importantly, studies on apo Delta(88-98)A-I(+his) provide the first experimental evidence that a native-like structure is not necessary for native-like lipid affinity, but apparently is necessary for both DMPC solubilization and LCAT activation. These results provide support for a hypothetical, multistep structure-based mechanism for apo hA-I lipid binding.

Apolipoprotein A-I↗
PubMed1998

Structural analysis of apolipoprotein A-I: effects of amino- and carboxy-terminal deletions on the lipid-free structure.

An amino-terminal deletion mutant (residues 1-43) and a carboxy-terminal deletion mutant (residues 187-243) of human apoliprotein A-I (apo hA-I) have been produced from a bacterial expression system to explore the importance of the missing residues for the conformation of apo hA-I. Our focus has been to study the lipid-free structure of apo hA-I to understand how discrete domains influence the conformational plasticity of the protein and, by inference, the mechanism of lipid binding. All spectral and physical measurements indicate that both apo delta(1-43)A-I and apo delta(187-243)A-I have folded, tertiary structures. These structures differ in the specific arrangement of helical domains based, in part, on their relative thermodynamic stability, near- and far-UV CD, limited proteolysis, and the accessibility of tryptophans to fluorescence quenchers. In addition, all data indicate that the folded domains of apo hA-I and apo delta(187-243)A-I are very similar. Results from analytical ultracentrifugation suggest that lipid-free apo hA-I and the deletion mutants each exist in a dynamic equilibrium between a loosely folded, helical bundle and an elongated monomeric helical hairpin. The conformational heterogeneity is consistent with significant ANS binding exhibited by all three proteins and could help to explain the facile lipid binding properties of apo hA-I.

Amino Acid Sequence↗
PubMed1997

The cyanobacterial repressor SmtB is predominantly a dimer and binds two Zn2+ ions per subunit.

The Synechococcus PCC7942 metallothionein repressor gene smtB has been cloned into a high expression vector and the protein purified to near homogeneity (>/=98%). Analytical ultracentrifugation studies demonstrate that the protein is predominantly dimeric in 0.1 M NaCl, pH 7.4, and 22 degrees C, exhibiting a monomer-dimer-tetramer equilibrium. The monomer-dimer (Ka(1,2)) and the dimer-tetramer (Ka(2,4)) association constants are 3.24 x 10(5) and 9.90 x 10(2) M-1, respectively. The repressor binds two Zn2+ ions per subunit with an overall Kd of 3.49 x 10(-6) M. In the presence of Zn2+, Ka(1, 2) increases by 2 orders of magnitude to 1.25 x 10(7) M-1 and the apparent weight-averaged sedimentation coefficient increases from 2. 00 to 2.22 S. The fact that the increase in sedimentation coefficient is greater than that predicted by increased dimerization is interpreted as caused by compaction of the structure in the presence of metal ions. At pH 6.0, 0.1 M NaCl, and 22 degrees C, the protein exhibits only a monomer-dimer equilibrium, with Ka(1,2) = 1.52 x 10(7) M-1 which is almost identical to that seen upon binding Zn2+ at pH 7.4. The compaction and conformational change in SmtB caused by Zn2+ is consistent with a role for this altered quaternary state in derepression of smtA in Synechococcus challenged with heavy metal ions.

Bacterial Proteins↗
PubMed1997

Relationship of visceral adipose tissue and glucose disposal is independent of sex in black NIDDM subjects.

To determine the interrelationship among insulin action, total or regional adiposity, and sex, we measured insulin-mediated glucose disposal by the euglycemic insulin clamp and adipose distribution using computed axial tomography (22 scans) in 32 black men and 20 black women with non-insulin-dependent diabetes mellitus (age 48 +/- 9 and 54 +/- 9 yr, body mass index 26.3 +/- 2.3 and 27.2 +/- 2.6 kg/m2, respectively). Women had approximately 80% more total and subcutaneous fat volume than men (31.8 +/- 8.3 vs. 18.6 +/- 6.1 and 28.5 +/- 7.3 vs. 14.7 +/- 4.6 liters) and less muscle volume (22.9 +/- 3.7 vs. 35.1 +/- 3.8 liters). Visceral fat volume did not differ between men and women (3.49 +/- 1.65 vs. 2.96 +/- 1.22 liters). Despite these body composition differences, an inverse nonlinear relationship existed between glucose disposal and visceral fat independent of sex (r = -0.58, P < 0.0001; men r = -0.60 and women r = -0.59; the slope and intercept were not different in men and women). Visceral fat explained a significant portion (34%) of variance in insulin-mediated glucose disposal, whereas total or subcutaneous fat and sex did not. Visceral fat appears to affect glucose disposal over a restricted range (up to approximately 2.5 l/m2 body surface area.

Adipose Tissue↗
PubMed1995

Oligomerization of the hydrophobic heptad repeat of gp41.

The transmembrane protein of human immunodeficiency virus type 1 (HIV-1) contains a leucine zipper-like (hydrophobic heptad) repeat which has been predicted to form an amphipathic alpha helix. To evaluate the potential of the hydrophobic heptad repeat to induce protein oligomerization, this region of gp41 has been cloned into the bacterial expression vector pRIT2T. The resulting plasmid, pRIT3, expresses a fusion protein consisting of the Fc binding domain of monomeric protein A, a bacterial protein, and amino acids 538 to 593 of HIV-1 gp41. Gel filtration chromatography demonstrated the presence of oligomeric forms of the fusion protein, and analytical centrifugation studies confirmed that the chimeric protein formed a higher-order multimer that was greater than a dimer. Thus, we have identified a region of HIV-1 gp41 which is capable of directing the oligomerization of a fusion protein containing monomeric protein A. Point mutations, previously shown to inhibit the biological activity of the HIV-1 envelope glycoprotein, have been engineered into the segment of gp41 contained in the fusion protein, and expressed mutant proteins were purified and analyzed via fast protein liquid chromatography. A point mutation in the heptad repeat, which changed the central isoleucine to an alanine, caused a significant (> 60%) decrease in oligomerization, whereas changing the central isoleucine to aspartate or proline resulted in almost a complete loss of oligomerization. Deletions of one, two, or three amino acids following the first isoleucine also resulted in a profound decrease in oligomerization. The inhibitory effects of the mutations on oligomer formation correlated with the effects of the same mutations on envelope glycoprotein-mediated fusion. A possible role of the leucine zipper-like region in the fusion process and in an oligomerization event distinct from assembly of the envelope glycoprotein complex is discussed.

Amino Acid Sequence↗
PubMed1994

Structural and kinetic studies of the 10 S<==>6 S transition in smooth muscle myosin.

The conformational transitions that smooth muscle myosin undergoes after nucleotide binding have been examined using fluorescently labeled nucleotides and regulatory light chain. The 10 S conformation of smooth muscle myosin could be induced by addition of 1-N6-ethenoadenosine or mant ADP plus beryllium fluoride, as well as by mant adenosine 5'-(beta,gamma-iminotriphosphate) (AMPPNP). Fluorescence lifetime studies using 1-N6-ethenoadenosine plus beryllium fluoride reveal two components for both (10 S)- and (6 S)-myosins, with little difference in the values of these lifetimes, their fractional amplitudes, or solute accessibilities. Anisotropy decay studies of myosin-mant nucleotide complexes demonstrate that the rotational correlation time for (10 S)-myosin is nearly 4-fold longer than that for (6 S)-myosin. Qualitatively similar results were obtained with a 5-[[[2(iodoacetyl)amino]ethyl]amino]naphthalene-1-sulfonic acid fluorescent probe attached to the regulatory light chain. Mant AMPPNP can be trapped in the active site by (10S)-myosin. Actin accelerates this release rate by 40-50-fold. These studies reveal: 1) reduction in nucleotide release rate by converting (6S) to (10S)-myosin is not due to a reduction in solute accessibility of the nucleotide 2) the heads in (10 S)-myosin are rigidly attached to the rest of the molecule, while in (6 S)-myosin, they have segmental flexibility, 3) regulatory light chain phosphorylation mimics the effect of high salt in enhancing segmental flexibility of the myosin heads, and 4) actin can induce the unfolding of (10 S)-myosin in the absence of regulatory light chain phosphorylation.

Acrylamide↗